CN1214824C - Method of regulating degradation rate of porous collagen-based cradle with amino acid - Google Patents
Method of regulating degradation rate of porous collagen-based cradle with amino acid Download PDFInfo
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- CN1214824C CN1214824C CN 03117083 CN03117083A CN1214824C CN 1214824 C CN1214824 C CN 1214824C CN 03117083 CN03117083 CN 03117083 CN 03117083 A CN03117083 A CN 03117083A CN 1214824 C CN1214824 C CN 1214824C
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Abstract
The present invention discloses a method for regulating and controlling the degradation rate of a collagen base porous stent by amino acid. Amino acid with good biocompatibility is used as a crosslinking and bridging agent by the method, amino acid of 0.1 to 100 muM is compounded with the collagen base porous stent so that a carbodiimide class compound is adopted for crosslinking treatment, and the collagen base porous stent with a controllable degradation rate and good biocompatibility is prepared. The method of the present invention has the advantages of no toxic or side effect of used materials, wide sources, low cost, simple and feasible preparation technology, mild conditions and good repeatability. The obtained porous stent can satisfy different requirements of tissue and organ regeneration, can be widely applied to the regeneration and the reconstruction of organs, such as skin, cartilage, bones, blood vessels, nerves, tendons, heart valves, etc., and has wide application value in the fields of biomedicine, tissue engineering, etc.
Description
Technical field
The present invention relates to aminoacid is crosslinked bridging agent, the method for regulation and control hole, collagen Quito scaffold degradation speed under the effect of cross-linking agent.
Background technology
It is multidisciplinary, multi-field that organizational project relates to medical science, chemistry, biology, materialogy etc.In the research of organizational project, the selection of timbering material and the structure of support are key links wherein.Collagen is the main component in the mammal connective tissue, constitutes the protein of human body about 30%, and the dry weight in skin reaches 72%.Collagen has 19 types, and modal is I type, II type and III type.Wherein the I type is the abundantest, and function admirable, is widely used in biomaterial.Though collagen-based materials has unrivaled biocompatibility, the mechanical strength of the support that makes up with pure collagen is lower, and degradation rate is too fast, can not satisfy the requirement of tissue engineering bracket.Therefore be necessary by suitable method crosslinked improving the degree of cross linking of hole, collagen Quito support, and the degradation rate that is complementary of acquisition and tissue regeneration.At present, the cross-linking method of hole, collagen Quito support mainly contains physical crosslinking and chemical crosslinking.The physical crosslinking method mainly contains xeothermic crosslinked, ultraviolet-crosslinkable of vacuum etc.The characteristics of these class methods are not introduce other material in cross-linking process, can not destroy hole, collagen Quito support excellent biological compatibility, but simple physical crosslinking can not reach the desired degree of cross linking, and the degradation rate of collagen scaffold is still too fast, can not be complementary with the regeneration rate of tissue.Chemical crosslink technique is used more widely, and chemical cross-linking agent mainly contains glutaraldehyde, Carbodiimides and bis-epoxy compounds.The shortcoming of traditional pure chemistry cross-linking method is relatively more difficult usually for the regulation and control of the degree of cross linking, can not satisfy the requirement that different various tissues of regeneration rate and organ make up; During with glutaraldehyde as cross linker, the residual meeting of glutaraldehyde causes some side effect, as cytotoxicity.Aminoacid is proteinic basic structural unit, has excellent biological compatibility.And have two carboxyls or two amino in acidic amino acid or the basic amino acid molecule respectively, therefore in collagen base support crosslinked, can be used as crosslinked bridging agent.
Summary of the invention
The purpose of this invention is to provide a kind of method of regulating and control hole, collagen Quito scaffold degradation speed with aminoacid as crosslinked bridging agent.
Method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed of the present invention, its step is earlier hole, collagen Quito to be propped up to be placed in the Freamine that concentration is 0.1~100 μ M to soak 0.01~100 hour, then with compound hole, amino acid whose collagen Quito support in 1~1000mM carbodiimides compounds, carrying out crosslinked 1~48 hour under 0~100 ℃, get final product.
By changing amino acid whose kind, the concentration of amino acid whose ratio and cross-linking agent can be regulated and control the degradation rate of hole, collagen Quito support.
Among the present invention, the aminoacid kind is not had special requirement, adopt lysine, arginine, glutamic acid, aspartic acid etc. usually.The carbodiimides compounds is not had special requirement yet, but preferably adopt water-soluble carbodiimide, as 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides (EDAC).Hole, collagen Quito support is meant pure collagen or the compound porous support of other composition, as compound aminoacid, and polysaccharide compound or the compound porous support of somatomedin compounds, said polysaccharide compound is as chitosan, chondroitin sulfate.The somatomedin compounds is as bone morphogenetic protein, fibroblast growth factor, endothelial cell growth factor (ECGF), transforming growth factor etc.Said collagen source is not had special requirement, can be beef tendon collagen, pigskin collagen or Mus tail collagen etc.
The used crosslinked bridging agent of the inventive method has excellent biological compatibility and degradation property, has no side effect, and wide material sources, and with low cost, preparation is simple, mild condition, good reproducibility.Invention is crosslinked bridging agent with aminoacid, by changing amino acid whose kind, the concentration of amino acid whose ratio and cross-linking agent realizes the degradation rate of regulation and control hole, collagen Quito support, the porous support of gained can satisfy the needs of different tissues and neomorph, can be widely used in the regeneration and the reconstruction of organs such as skin, cartilage, bone, blood vessel, nerve, tendon and cardiac valve, be with a wide range of applications in fields such as biomedical and organizational projects.
Description of drawings
Fig. 1 is for adding the influence of variety classes aminoacid to hole, collagen Quito scaffold degradation degree.Support the type i collagen enzymatic solution of 0.1mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 2 is the variation relation of the degree of degradation of hole, collagen Quito support with aminoglutaric acid concentration.Support the type i collagen enzymatic solution of 0.5mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 3 is the variation relation of the degree of degradation of hole, collagen Quito support with lysine concentration.Support the type i collagen enzymatic solution of 0.5mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 4 is the variation relation of the palliating degradation degree of hole, collagen Quito support with 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides N-maloyl imines (EDAC/NHS) concentration.Support the type i collagen enzymatic solution of 0.1mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 5 is for the contrast collagen scaffold and add the collagen Quito hole support of 2.5 μ M lysines after crosslinked, the external contraction behavior under the fibroblast effect.Cell inoculation density is 500 * 10
4/ ml.
Fig. 6 is for adding the crosslinked laser confocal microscope photo of collagen scaffold (rhodamine labelling) plantation fibroblast after 3 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml, fluorescein(e) diacetate (FDA) dyeing.
Fig. 7 a is for adding crosslinked scanning electron microscope (SEM) photo of collagen scaffold plantation fibroblast after 7 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml.
Fig. 7 b is for adding the crosslinked SEM photo of collagen scaffold plantation fibroblast after 21 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml.
Fig. 8 is for adding the crosslinked tissue slice photo (H﹠amp of collagen scaffold plantation fibroblast after 21 days of 2.5 μ M lysines; E dyeing).Cell inoculation density is 500 * 10
4/ ml.
The specific embodiment
The present invention may be better understood by following embodiment, but these examples are not used for limiting the present invention.
Different types of aminoacid is to the influence of the degree of degradation of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add 2-N-morpholino ethane sulfonic acid (MES) solution (50mM of glycine (Gly), glutamic acid (Glu) or lysine (Lys) that 1ml contains 2.5 μ M respectively, pH is 5.5), soaked 1 hour; Soak in contrast to add tri-distilled water simultaneously.Add 1ml then respectively and contain 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides (EDAC) of 40mM, the MES solution of the N-maloyl imines (NHS) of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain hole, the collagen Quito support after crosslinked.
Get uncrosslinked, do not add aminoacid crosslinked, add crosslinked hole, the collagen Quito support of different aminoacids, (PBS pH7.4), digests 12h in 37 ℃ of thermostatic water bath to add the type i collagen enzymatic solution of 0.1mg/ml of 3ml.Draw digestion supernatant 1ml, place polymerization pipe, add the hydrochloric acid of the 6M of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.Degree of degradation uncrosslinked, that do not add the porous support that aminoacid is crosslinked, the interpolation different aminoacids is crosslinked is seen Fig. 1.
The concentration of glutamic acid is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of 1~10 μ M glutamic acid of 1ml respectively, soaked 1 hour; Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain hole, the collagen Quito support after crosslinked.
Get and add the crosslinked porous support of variable concentrations glutamic acid, (PBS pH7.4), digests 12h to the type i collagen enzymatic solution of the 0.5mg/ml of adding 3ml in 37 ℃ of thermostatic water bath.Draw digestion supernatant 1ml, place polymerization pipe, add the 6M hydrochloric acid of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of glutamic acid is seen Fig. 2 to the influence of the degree of degradation of hole, collagen Quito support.
Embodiment 3
The concentration of lysine is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of 0.67~100 μ M lysine of 1ml respectively, soaked 1 hour; Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get and add the crosslinked porous support of variable concentrations lysine, (PBS pH7.4), digests 12h to the type i collagen enzymatic solution of the 0.5mg/ml of adding 3ml in 37 ℃ of thermostatic water bath.Draw digestion supernatant 1ml, place polymerization pipe, add the 6M hydrochloric acid of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of lysine is seen Fig. 3 to the influence of the degree of degradation of hole, collagen Quito support.
Embodiment 4
The cross-linking agent of variable concentrations is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of the lysine of 2.5 μ M respectively, soaked 1 hour; (50/25~5/2.5mM) EDAC/NHS solution, under room temperature crosslinked 24 hours, crosslinked back was with tri-distilled water rinsing 6 times, each 10 minutes to add the variable concentrations of 1ml then respectively.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get the three-dimensional rack 2.5mg after crosslinked, the type i collagen enzymatic solution (being made into PBS) that adds the 0.1mg/ml of 3ml then digests in 37 ℃ of thermostatic water bath, to required time absorption digestion supernatant 1ml, place polymerization pipe, the 6M hydrochloric acid that adds 2ml, hydrolysis 12 hours in 120 ℃ baking oven behind the tube sealing.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of cross-linking agent is seen Fig. 4 to the influence of the degree of degradation of hole, collagen Quito support.
The interpolation of lysine is to the influence of the external contraction behavior of hole, collagen Quito support under the fibroblast effect.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of lysine of the 2.5 μ M of 1ml respectively, soaked 1 hour, do not add amino acid whose sample with corresponding M ES solution soaking 1 hour.Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get uncrosslinked, do not add aminoacid crosslinked, add the crosslinked three-dimensional rack of lysine, plant human fibroblasts (5,000,000/ml), 37 ℃ of CO of 5% down of 400 μ l respectively
2Cultivate in the incubator, the next day change liquid.After having cultivated 3,7,14 and 21 days, measure the diameter of various supports respectively.Fig. 5 is the external contraction behaviors of various supports under the fibroblast effect.
Add the cell in vitro evaluation of hole, the collagen Quito support of lysine
The collagen scaffold (2.5mg/ piece) of getting the rhodamine labelling places 24 well culture plates, adds the MES solution (50mM, pH are 5.5) of the lysine of 1ml concentration 2.5 μ M respectively, soaks 1 hour.Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through freezing-lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get and add the crosslinked collagen porous support of lysine, plant human fibroblasts (5% the CO under 5,000,000/ml), 37 ℃ of 400 μ l
2Cultivate in the incubator, the next day change liquid.After having cultivated 3 days, use fluorescein diacetate (FDA) dyeing back laser confocal microscope (CLSM) and observe respectively.As seen from Figure 6, fibroblast (left side) the plantation 3 days after can along collagen scaffold (in) hole wall sprawl, present good growth conditions (right side).Fig. 7 a and Fig. 7 b are respectively scanning electron microscope (SEM) photo of cultivating after 7 days and 21 days.Visible fibroblast is attached on the collagen and sprawls among the figure, presents typical fusiformis.Fig. 8 as seen from the figure at internal stent, has a large amount of fibroblasts to grow into, and is evenly distributed in support everywhere for the tissue slice figure of the support of cultivation after 21 days.
Claims (5)
1. method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed, it is characterized in that earlier hole, collagen Quito being propped up to be placed in the Freamine that concentration is 0.1~100 μ M soaking 0.01~100 hour, then compound hole, amino acid whose collagen Quito support was being carried out in 1~1000mM carbodiimides compounds crosslinked 1~48 hour under 0~100 ℃.
2. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that said aminoacid is lysine, arginine, glutamic acid, glycine or aspartic acid.
3. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that hole, said collagen Quito support is meant pure collagen porous support or compound hole, the collagen Quito support of aminoacid, polysaccharide compound or somatomedin compounds.
4. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 3, it is characterized in that polysaccharide compound is chitosan or chondroitin sulfate, the somatomedin compounds is bone morphogenetic protein, fibroblast growth factor, endothelial cell growth factor (ECGF) or transforming growth factor.
5. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that said carbodiimides compounds is a water-soluble carbodiimide.
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